In-situ entrapment of laccase in mesoporous silica microparticles for degradation of oxyteracycline

Abstract

A simple and reproducible method for in-situ entrapment of laccase in mesoporous silica microparticles (LSM) was studied. This involved the hydrolysis and condensation of tetraethyl orthosilicate (TEOS) via sol-gel route using one-step (base catalyst) and two-step (acid-base catalyst) methods followed by an ambient drying procedure. It was found that the one-step method was not suitable for in-situ entrapment as it left a significant amount of untrapped laccase in the reaction media which led to the inactivation of laccase due to its active site alteration by continuous contact with basic condition. Conversely, the laccase was entrapped entirely in the silica matrices which were synthesized using the two-step method with the highest specific catalytic activity of 434.71 U/g obtained from the 2-LSM15 sample. In addition, the LSM showed an improvement in stability towards pH and temperature compared to the free laccase and was able to retain more than 80% of its initial catalytic activity after one month of storage. The synthesis condition for laccase entrapment was then optimized using a 3-level-4-factor Box–Behnken experimental design to investigate the relationships of the starting material compositions towards the catalytic activity of the entrapped laccase. The optimal condition for laccase entrapment obtained from the response surface methodology (RSM) at H2O/TEOS = 5.44 by molar, HCl = 2.52 mol ×10-6, TEA = 0.39 mol ×10-3 and Lac = 3.83 mg/ml. The predicted response of the maximum solution was 301.7 U/g and the experimental value was 298.36 U/g, respectively, under the optimal condition. Moreover, the sample was capable of retaining almost 90% of the original catalytic activity after 10 repeated recovery and uses. The application of the LSM was further investigated for the degradation of oxytetracycline (OTC). As the temperature increases, OTC component became unstable thus made the use of laccase for OTC degradation unnecessary. On the other hand, the OTC component turned out to be more stable as the pH increased. However, when LSM was applied, 68-88 % of OTC was degraded under previous circumstances. In the kinetic study, opposite pattern of the degradation kinetics rate constants was observed for free laccase and LSM as the amount of enzyme loading increases. The corresponding constant values for free laccase decreased, while the values for LSM experienced a decent escalation. The LSM with a dosage of 4:1 resulted in the highest turnover number (Kcat= 140136.99 min-1) of OTC molecules converted to product per enzyme molecule per unit of time and with catalytic efficiency, Kcat/Km= 814.75.